Terminal-phosphate-labeled nucleotides and methods of use

ABSTRACT

The present invention relates to improved methods of detecting a target using a labeled substrate or substrate analog. The methods comprise reacting the substrate or substrate analog in an enzyme-catalyzed reaction which produces a labeled moiety with independently detectable signal only when such substrate or substrate analog reacts. The present invention, in particular, describes methods of detecting a nucleic acid in a sample, based on the use of terminal-phosphate-labeled nucleotides as substrates for nucleic acid polymerases. The methods provided by this invention utilize a nucleoside polyphosphate, dideoxynucleoside polyphosphate, or deoxynucleoside polyphosphate analogue which has a colorimetric dye, chemiluminescent, or fluorescent moiety, a mass tag or an electrochemical tag attached to the terminal-phosphate. When a nucleic acid polymerase uses this analogue as a substrate, an enzyme-activatable label would be present on the inorganic polyphosphate by-product of phosphoryl transfer. Cleavage of the polyphosphate product of phosphoryl transfer via phosphatase leads to a detectable change in the label attached thereon. When the polymerase assay is performed in the presence of a phosphatase, there is provided a convenient method for real-time monitoring of DNA or RNA synthesis and detection of a target nucleic acid.

RELATED APPLICATIONS

This application is a continuation in part of U.S. patent applicationSer. No. 10/113,030 filed Apr. 1, 2002 now U.S. Pat. No. 7,052,839.

FIELD OF THE INVENTION

The present invention relates to improved methods of detecting a targetusing a labeled substrate or substrate analog. The improvement comprisesreacting the substrate or substrate analog in an enzyme-catalyzedreaction which produces a labeled moiety with independently detectablesignal only when such substrate or substrate analog reacts. The presentinvention particularly relates to methods of detecting a polynucleotidein a sample, based on the use of terminal-phosphate-labeled nucleotidesincluding three or more phosphates as substrates for nucleic acidpolymerases. The labels employed are dyes, which undergo a chemicalchange and become a fluorescent or color producing reagent only upon theaction of the polymerase.

BACKGROUND OF THE INVENTION

Methods are known for detecting specific nucleic acids or analytes in asample with high specificity and sensitivity. Such methods generallyrequire first amplifying nucleic acid sequence based on the presence ofa specific target sequence or analyte. Following amplification, theamplified sequences are detected and quantified. Conventional detectionsystems for nucleic acids include detection of fluorescent labels,fluorescent enzyme-linked detection systems, antibody-mediated labeldetection, and detection of radioactive labels.

One disadvantage of detection methods presently widely in use is theneed to separate labeled starting materials from a final labeled productor by-product. Such separations generally require gel electrophoresis orimmobilization of a target sequence onto a membrane for detection.Moreover, there are often numerous reagents and/or incubation stepsrequired for detection.

It has been known that DNA and RNA polymerases are able to recognize andutilize nucleosides with a modification at or in place of the gammaposition of the triphosphate moiety. It is further known that theability of various polymerases to recognize and utilize gamma-modifiednucleotide triphosphates (NTP's) appears to vary depending on the moietyattached to the gamma phosphate. In general, RNA polymerases are morepromiscuous than DNA polymerases.

A colorimetric assay for monitoring RNA synthesis from RNA polymerasesin presence of a gamma-phosphate modified nucleotide has been previouslyreported. In this prior report, RNA polymerase reactions were performedin the presence of a gamma-modified, alkaline phosphatase resistantnucleotide triphosphate which was modified at its gamma-phosphate with adinitrophenyl group. When RNA polymerase reactions were performed in thepresence of this gamma-modified NTP as the sole nucleotide triphosphateand a homopolymeric template, it was found that RNA polymerase couldrecognize and utilize the modified NTP. Moreover, when the polymerasereactions were performed in the presence of an alkaline phosphatase,which digested the p-nitrophenyl pyrophosphate aldo-product ofphosphoryl transfer to the chromogenic p-nitrophenylate, an increase inabsorbence was reported. A disadvantage of this detection method is thatthe real-time colorimetric assay, performed in the presence of analkaline phosphatase, only works with a homopolymeric template.

It would, therefore, be of benefit to provide a method for detecting RNAin the presence of a heteropolymeric template, which method would not berestricted to using a single terminal-phosphate modified nucleotide asthe sole nucleotide that is substantially non-reactive to alkalinephosphatase. This would allow for a single-tube assay for real-timemonitoring of RNA synthesis using hetero-polymeric templates.

Generally, the fluorescent dyes in the assays are quenched by a moleculeplaced in close proximity to them in the labeled entity, and thedetectable signal is produce when the structure is altered and thequencher is either removed, moved away from the dye, or otherwiserendered inactive. At that point a detectable signal is produced.However, since quenching is not absolute, the dynamic range of suchassays is limited.

It would further be of benefit to provide for similar assays for RNAwherein the identity of the label on the terminal-phosphate is varied toallow for better recognition and utilization by RNA polymerase.Furthermore, it is desired that the label on the terminal-phosphatecould be varied so as to allow for chemiluminescent and fluorescentdetection, or reduction potential, as well as for improved calorimetricdetection, wherein only simple and routine instrumentation would berequired for detection, and increased dynamic range.

Given that DNA polymerases are known in the art to be less promiscuousthan RNA polymerases regarding recognition and utilization ofterminally-modified nucleotides, wherein the identity of the moiety atthe terminal position can largely affect the DNA polymerase'sspecificity toward the nucleotide, it would be highly desired to providefor a non-radioactive method for detecting DNA by monitoring DNApolymerase activity. Furthermore, it would be desired that the synthesisand detection of DNA could be accomplished in a single-tube assay forreal-time monitoring and that the label at the terminal-phosphate ofnucleotide substrates could encompass chemiluminescent, fluorescent, andcalorimetric detection, as well as analysis by mass or reductionpotential.

SUMMARY OF THE INVENTION

The present invention relates to improved methods of detecting a targetusing a labeled substrate or substrate analog. The improvement comprisesreacting the substrate or substrate analog in an enzyme-catalyzedreaction which produces a labeled moiety with independently detectablesignal only when such substrate or substrate analog reacts. The presentinvention also provides for a method of detecting the presence of anucleic acid sequence including the steps of: a) conducting a nucleicacid polymerase reaction, wherein the reaction includes the reaction ofa terminal-phosphate-labeled nucleotide, which reaction results in theproduction of labeled polyphosphate; b) permitting the labeledpolyphosphate to react with a phosphatase to produce a detectablespecies; and c) detecting the presence of the detectable species. Adefinition of phosphatase in the current invention includes any enzymewhich cleaves phosphate mono esters, polyphosphates and nucleotides torelease inorganic phosphate. In the context of the present invention,this enzyme does not cleave a terminally labeled nucleoside phosphate(i.e. the terminal-phosphate-labeled nucleotide is substantiallynon-reactive to phosphatase). The phosphatase definition herein providedspecifically includes, but is not limited to, alkaline phosphatase (EC3.1.3.1) and acid phosphatase (EC 3.1.3.2). The definition of anucleotide in the current invention includes a natural or modifiednucleoside phosphate.

The invention further provides for a method of detecting the presence ofa DNA sequence including the steps of: a) conducting a DNA polymerasereaction in the presence of a terminal-phosphate-labeled nucleotide,which reaction results in the production of a labeled polyphosphate; b)permitting the labeled polyphosphate to react with a phosphatase toproduce a detectable species; and c) detecting the presence of thedetectable species.

Also provided is a method of detecting the presence of a nucleic acidsequence comprising the steps of: (a) conducting a nucleic acidpolymerase reaction in the presence of at least oneterminal-phosphate-labeled nucleotide having four or more phosphategroups in the polyphosphate chain, which reaction results in theproduction of a labeled polyphosphate; and (b)detecting the labeledpolyphosphate.

In addition, the invention relates to a method of detecting the presenceof a nucleic acid sequence comprising the steps of: (a) conducting anucleic acid polymerase reaction in the presence of at least oneterminal-phosphate-labeled nucleotide having four or more phosphategroups in the polyphosphate chain, which reaction results in theproduction of a labeled polyphosphate; (b) permitting the labeledpolyphosphate to react with a phosphatase to produce a detectablespecies; and (c) detecting the presence of the detectable species.

A further aspect of the present invention relates to a method ofquantifying a nucleic acid including the steps of: (a) conducting anucleic acid polymerase reaction, wherein the reaction includes thereaction of a terminal-phosphate-labeled nucleotide, which reactionresults in production of labeled polyphosphate; (b) permitting thelabeled polyphosphate to react with a phosphatase to produce adetectable by-product species in an amount substantially proportional tothe amount of nucleic acid; (c) measuring the detectable species; and(d) comparing the measurements using known standards to determine thequantity of nucleic acid.

The invention further relates to a method of quantifying a DNA sequenceincluding the steps of: (a) conducting a DNA polymerase reaction in thepresence of a terminal-phosphate-labeled nucleotide, the reactionresulting in production of labeled polyphosphate; (b) permitting thelabeled polyphosphate to react with a phosphatase to produce adetectable by-product species in amounts substantially proportional tothe amount of the DNA sequence; (c)measuring the detectable species; and(d) comparing the measurements using known standards to determine thequantity of DNA.

Another aspect of the invention relates to a method for determining theidentity of a single nucleotide in a nucleic acid sequence, whichincludes the steps of: (a) conducting a nucleic acid polymerase reactionin the presence of at least one terminal phosphate-labeled nucleotide,which reaction results in the production of labeled polyphosphate; (b)permitting the labeled polyphosphate to react with a phosphatase toproduce a detectable species; (c) detecting the presence of thedetectable species; and (d) identifying the nucleoside incorporated.

Also provided is a method for determining the identify of a singlenucleotide in a nucleic acid sequence including the following steps: (a)conducting a nucleic acid polymeric reaction in the presence of at leastone terminal-phosphate-labeled nucleotide having four or more phosphategroups in the polyphosphate chain, which reaction results in theproduction of labeled polyphosphate; (b) permitting the labeledpolyphosphate to react with a phosphatase to produce a detectablespecies; (c) detecting the presence of said detectable species; and (d)identifying the nucleoside incorporated.

The present invention further includes a nucleic acid detection kitwherein the kit includes:

(a) at least one or more terminal-phosphate-labeled nucleotide accordingto Formula I below:

wherein P=phosphate (PO₃) and derivatives thereof, n is 2 or greater; Yis an oxygen or sulfur atom; B is a nitrogen-containing heterocyclicbase; S is an acyclic moiety, carbocyclic moiety or sugar moiety; L isan enzyme-activatable label containing a hydroxyl group, a sulfhydrylgroup or an amino group suitable for forming a phosphate ester, athioester or a phosphoramidate linkage at the terminal phosphate of anatural or modified nucleotide; P-L is a phosphorylated label whichpreferably becomes independently detectable when the phosphate isremoved.

(b) at least one of DNA polymerase, RNA polymerase, or reversetranscriptase; and

(c) phosphatase.

The invention further provides for a process whereby the assay utilizesa phosphate labeled nucleotide in which the label is not detectableuntil the base is incorporated into the nucleotide, releasing thelabeled pyrophosphate, which can then be detected by the action of asuitable enzyme such as a phosphatase. Since the label is not detectableunless the base is incorporated, the assay can be run in a homogeneousformat, or in a single vessel which does not require multiple additionof reagents. Additionally, no separation of unreacted reagents will berequired because the label in unreacted nucleotides will produce nosignal. Further, because the signal will only be generated if the baseis incorporated, the system will have a large dynamic range and, if twoor more, preferably four, different labeled nucleotides are used, theassay can be multiplexed.

Further provided are new compositions comprising dye polyphosphates offormula:Dye-(P)_(n)-P

Wherein, when n is 2-5, the dye is selected from the group consisting ofa fluorescent, colored, fluorogenic, chromogenic, luminogenic dye or anelectrochemical label. when n is 1-5, the dye is a fluorogenic or aluminogenic moiety or an electrochemical label.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing fluorescence obtained by polymeraseutilization of a gamma-phosphate-labeled ddGTP in a template-directedprocess in the presence of phosphatase.

FIG. 2 is a graph showing fluorescence obtained by polymeraseutilization of a gamma-phosphate-labeled ddATP in a template-directedprocess in the presence of phosphatase.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The term “nucleoside” as defined herein is a compound including a purinedeazapurine, pyrimidine or modified base linked to a sugar or a sugarsubstitute, such as a carbocyclic or acyclic moiety, at the 1′ positionor equivalent position and includes 2′-deoxy and 2′-hydroxyl, and 2′,3′-dideoxy forms as well as other substitutions.

The term “nucleotide” as used herein refers to a phosphate ester of anucleoside, wherein the esterification site typically corresponds to thehydroxyl group attached to the C-5 position of the pentose sugar.

The term “oligonucleotide” includes linear oligomers of nucleotides orderivatives thereof, including deoxyribonucleosides, ribonucleosides,and the like. Throughout the specification, whenever an oligonucleotideis represented by a sequence of letters, the nucleotides are in the5′→3′ order from left to right where A denotes deoxyadenosine, C denotesdeoxycytidine, G denotes deoxyguanosine, and T denotes thymidine, unlessnoted otherwise.

The term “primer” refers to a linear oligonucleotide that anneals in aspecific way to a unique nucleic acid sequence and allows foramplification of that unique sequence.

The phrase “target nucleic acid sequence” and the like refers to anucleic acid whose sequence identity, or ordering or location ofnucleosides is determined by one or more of the methods of the presentinvention.

The term polyphosphate refers to two or more phosphates.

The term luminogenic moiety refers to a chemical moiety which produces achemiluminescent signal only upon activation.

The present invention relates to methods of detecting a polynucleotidein a sample wherein a convenient assay is used for monitoring RNA or DNAsynthesis via nucleic acid polymerase activity. RNA and DNA polymerasessynthesize oligonucleotides via transfer of a nucleoside monophosphatefrom a nucleoside triphosphate (NTP) or deoxynucleoside triphosphate(dNTP) to the 3′ hydroxyl of a growing oligonucleotide chain. The forcewhich drives this reaction is the cleavage of an anhydride bond and thecon-commitant formation of an inorganic pyrophosphate. The presentinvention utilizes the finding that structural modification of theterminal-phosphate of the nucleotide does not abolish its ability tofunction in the polymerase reaction. The oligonucleotide synthesisreaction involves direct changes only at the α- and β-phosphoryl groupsof the nucleotide, allowing nucleotides with modifications at theterminal phosphate position to be valuable as substrates for nucleicacid polymerase reactions.

In certain embodiments, the polymerase is a DNA polymerase, such as DNApolymerase I, II, or III or DNA polymerase α, β, γ, or terminaldeoxynucleotidyl transferase or telomerase. In other embodiments,suitable polymerases include, but are not limited to, a DNA dependentRNA polymerase, a primase, or an RNA dependant DNA polymerase (reversetranscriptase).

The methods provided by this invention utilize a nucleosidepolyphosphate, such as a deoxynucleoside polyphosphate,dideoxynucleoside polyphosphate, carbocyclic nucleoside polyphosphate,or acrylic nucleoside polyphosphate analogue with an electrochemicallabel, mass tag, or a colorimetric dye, chemiluminescent, or fluorescentlabel attached to the terminal-phosphate. When a nucleic acid polymeraseuses this analogue as a substrate, an enzyme-activatable label would bepresent on the inorganic polyphosphate by-product of phosphoryltransfer. Cleavage of the polyphosphate product of phosphoryl transfervia phosphatase, leads to a detectable change in the label attachedthereon. It is noted that while RNA and DNA polymerases are able torecognize nucleotides with modified terminal phosphoryl groups, theinventors have determined that this starting material is not a templatefor phosphatases. The scheme below shows the most relevant molecules inthe methods of this invention; namely the terminal-phosphate-labelednucleotide, the labeled polyphosphate by-product and theenzyme-activated label.

In the scheme above, n is 1 or greater, R₁ and R₂ are independently H,OH, SH, SR, OR, F, Br, Cl, I, N₃, NHR or NH₂; B is a nucleotide base ormodified heterocyclic base; X is O, S, or NH; Y is O, S, or BH₃; and Lis a phosphatase activatable label which may be a chromogenic,fluorogenic, chemiluminescent molecule, mass tag or electrochemical tag.A mass tag is a small molecular weight moiety suitable for massspectrometry that is readily distinguishable from other components dueto a difference in mass. An electrochemical tag is an easily oxidizableor reducible species. It has been discovered that when n is 2 orgreater, the nucleotides are significantly better substrates forpolymerases than when n is 1. Therefore, in preferred embodiments, n is2, 3 or 4, R₁ and R₂ are independently H or OH; X and Y are O; B is anucleotide base and L is a label which may be a chromogenic, fluorogenicor a chemiluminescent molecule.

In one embodiment of the method of detecting the presence of a nucleicacid sequence provided herein, the steps include (a) conducting anucleic acid polymerase reaction wherein the reaction includes aterminal-phosphate-labeled nucleotide wherein the polymerase reactionresults in the production of labeled polyphosphate; (b) permitting thelabeled polyphosphate to react with a phosphatase suitable to hydrolyzethe phosphate ester and to produce a detectable species; and c)detecting the presence of a detectable species by suitable means. Inthis embodiment, the template used for the nucleic acid polymerasereaction may be a heteropolymeric or homopolymeric template. Byterminal-phosphate-labeled nucleotide, it is meant throughout thespecification that the labeled polyphosphate con-committantly releasedfollowing incorporation of the nucleoside monophosphate into the growingnucleotide chain, may be reacted with the phosphatase to produce adetectable species. Other nucleotides included in the reaction which aresubstantially non-reactive to phosphatase may be, for example, blockedat the terminal-phosphate by a moiety which does not lead to theproduction of a detectable species. The nucleic acid for detection inthis particular embodiment may include RNA, a natural or syntheticoligonucleotide, mitochondrial or chromosomal DNA.

The invention further provides a method of detecting the presence of aDNA sequence including the steps of (a) conducting a DNA polymerasereaction in the presence of a terminal-phosphate labeled nucleotide,which reaction results in the production of a labeled polyphosphate; (b)permitting the labeled polyphosphate to react with a phosphatase toproduce a detectable species; and (c) detecting the presence of saiddetectable species. The DNA sequence for detection may include DNAisolated from cells, chemically treated DNA such as bisulfite treatedmethylated DNA or DNA chemically or enzymatically synthesized accordingto methods known in the art. Such methods include PCR, and thosedescribed in DNA Structure Part A: Synthesis and Physical analysis ofDNA, Lilley, D. M. J. and Dahlberg, J. E. (Eds.), Methods Enzymol., 211,Academic Press, Inc., New York (1992), which is herein incorporated byreference. The DNA sequence may further include chromosomal DNA andnatural or synthetic oligonucleotides. The DNA may be either double- orsingle-stranded.

The methods of the invention may further include the step of includingone or more additional detection reagents in the polymerase reaction.The additional detection reagent may be capable of a response that isdetectably different from the detectable species. For example, theadditional detection reagent may be an antibody.

Suitable nucleotides for addition as substrates in the polymerasereaction include nucleoside polyphosphates, such as including, but notlimited to, deoxyribonucleoside polyphosphates, ribonucleosidepolyphosphates, dideoxynucleoside polyphosphates, carbocyclic nucleosidepolyphosphates and acyclic nucleoside polyphosphates and analogsthereof. Particularly desired are nucleotides containing 3, 4, or 5phosphate groups in the polyphosphate chain, where the terminalphosphate is labeled.

It is noted that in embodiments including terminal-phosphate-labelednucleotides having four or more phosphates in the polyphosphate chain,it is within the contemplation of the present invention that the labeledpolyphosphate by-product of phosphoryl transfer may be detected withoutthe use of phosphatase treatment. For example, it is known that naturalor modified nucleoside bases, particularly guanine, can cause quenchingof fluorescent markers. Therefore, in a terminal-phosphate-labelednucleotide, the label may be partially quenched by the base. Uponincorporation of the nucleoside monophosphate, the label polyphosphateby-product may be detected due to its enhanced fluorescence.Alternatively, it is possible to physically separate the labeledpolyphosphate product by chromatographic separation methods beforeidentification by fluorescence, color, chemiluminescence, orelectrochemical detection. In addition, mass spectrometry could be usedto detect the products by mass difference.

The methods of the present invention may include conducting thepolymerase reaction in the presence of at least one of DNA or RNApolymerase. Suitable nucleic acid polymerases may also include primases,telomerases, terminal deoxynucleotidyl transferases, and reversetranscriptases. A nucleic acid template may be required for thepolymerase reaction to take place and may be added to the polymerasereaction solution. It is anticipated that all of the steps (a), (b) and(c) in the detection methods of the present invention could be runconcurrently using a single, homogenous reaction mixture, as well as runsequentially.

It is well within the contemplation of the present invention thatnucleic acid polymerase reactions may include amplification methods thatutilize polymerases. Examples of such methods include polymerase chainreaction (PCR), rolling circle amplification (RCA), and nucleic acidsequence based amplification (NASBA). For e.g., wherein the targetmolecule is a nucleic acid polymer such as DNA, it may be detected byPCR incorporation of a gamma-phosphate labeled nucleotide base such asadenine, thymine, cytosine, guanine or other nitrogen heterocyclic basesinto the DNA molecule. The polymerase chain reaction (PCR) method isdescribed by Saiki et al in Science Vol. 239, page 487, 1988, Mullis etal in U.S. Pat. No. 4,683,195 and by Sambrook, J. et al. (Eds.),Molecular Cloning, second edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1980), Ausubel, F. M. et al. (Eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., NY (1999), andWu, R. (Ed.), Recombinant DNA Methodology II, Methods in Zumulogy,Academic Press, Inc., NY, (1995). Using PCR, the target nucleic acid fordetection such as DNA is amplified by placing it directly into areaction vessel containing the PCR reagents and appropriate primers.Typically, a primer is selected which is complimentary in sequence to atleast a portion of the target nucleic acid.

It is noted that nucleic acid polymerase reactions suitable forconducting step (a) of the methods of the present invention may furtherinclude various RCA methods of amplifying nucleic acid sequences. Forexample, those disclosed in U.S. Pat. No. 5,854,033 to Lizardi, Paul M.,incorporated herein by reference, are useful. Polymerase reactions mayfurther include the nucleic acid sequence based amplification (NASBA)wherein the system involves amplification of RNA, not DNA, and theamplification is iso-thermal, taking place at one temperature (41° C.).Amplification of target RNA by NASBA involves the coordinated activitiesof three enzymes: reverse transcriptase, Rnase H, and T7 RNA polymerasealong with oligonucleotide primers directed toward the sample targetRNA. These enzymes catalyze the exponential amplification of a targetsingle-stranded RNA in four steps: extension, degradation, DNA synthesisand cyclic RNA amplification.

Methods of RT-PCR, RCA, and NASBA generally require that the originalamount of target nucleic acid is indirectly measured by quantificationof the amplification products. Amplification products are typicallyfirst separated from starting materials via electrophoresis on anagarose gel to confirm a successful amplification and are thenquantified using any of the conventional detection systems for a nucleicacid such as detection of fluorescent labels, enzyme-linked detectionsystems, antibody-mediated label detection and detection of radioactivelabels. In contrast, the present method eliminates the need to separateproducts of the polymerase reaction from starting materials before beingable to detect these products. For example, in the present invention, areporter molecule (fluorescent, chemiluminescent or a chromophore) orother useful molecule is attached to the nucleotide in such a way thatit is undetectable under certain conditions when masked by the phosphateattachment. However, following the incorporation of the nucleotide intothe growing oligonucleotide chain and phosphatase treatment of thereaction, the label is detectable under those conditions. For example,if the hydroxyl group on the side of the triple ring structure of1,3-dichloro-9,9-dimethyl-acridine-2-one (DDAO) is attached to theterminal-phosphate position of the nucleotide, the DDAO does notfluoresce at 659 nm. Once the nucleoside monophosphate is incorporatedinto DNA, the other product, DDAO polyphosphate (which also does notfluoresce at 659 nm) is a substrate for phosphatase. Oncede-phosphorylated to form DDAO, the dye moiety will become fluorescentat 659 nm and hence detectable. The specific analysis of thepolyphosphate product can be carried out in the polymerase reactionsolution, eliminating the need to separate reaction products fromstarting materials. This scheme allows for the detection and,optionally, quantitation of nucleic acids formed during polymerasereactions using routine instrumentation such as spectrophotometers.

In the methods described above, the polymerase reaction step may furtherinclude conducting the polymerase reaction in the presence of aphosphatase, which converts labeled polyphosphate by-product to thedetectable label. As such, a convenient assay is established fordetecting the presence of a nucleic acid sequence that allows forcontinuous monitoring of detectable species formation. This represents ahomogeneous assay format in that it can be performed in a single tube.

One format of the assay methods described above may include, but is notlimited to, conducting the polymerase reaction in the presence of asingle type of terminal-phosphate-labeled nucleotide capable ofproducing a detectable species, for example terminal-phosphate-modifiedATP, wherein all other nucleotides are substantially non-reactive tophosphatase, but yield non-detectable species.

In another assay format, the polymerase reaction may be conducted in thepresence of more than one type of terminal-phosphate-labeled nucleotide,each type capable of producing a uniquely detectable species. Forexample, the assay may include a first nucleotide (e.g., adenosinepolyphosphate) that is associated with a first label which whenliberated enzymatically from the inorganic polyphosphate by-product ofphosphoryl transfer, emits light at a first wavelength and a secondnucleotide (e.g., guanosine polyphosphate) associated with a secondlabel that emits light at a second wavelength. Desirably, the first andsecond wavelength emissions have substantially little or no overlap. Itis within the contemplation of the present invention that multiplesimultaneous assays based on nucleotide sequence information canthereafter be derived based on the particular label released from thepolyphosphate.

In one aspect of the methods of detecting the presence of a nucleic acidsequence described above, the terminal-phosphate-labeled nucleotide maybe represented by the following structure (Formula I):

wherein P=phosphate (PO₃) and derivatives thereof, n is 2 or greater; Yis an oxygen or sulfur atom; B is a nitrogen-containing heterocyclicbase; S is an acyclic moiety, carbocyclic moiety or sugar moiety; L isan enzyme-activatable label containing a hydroxyl group, a sulfhydrylgroup or an amino group suitable for forming a phosphate ester, athioester or a phosphoramidate linkage at the terminal phosphate of anatural or modified nucleotide; P-L is a phosphorylated label whichpreferably becomes independently detectable when the phosphate isremoved.

In addition to phosphate hydrolysis, many other kinds of reactions canbe chromogenic, fluorogenic, or result in the production ofindependently detectable labels. For example, glycosidases can act onchromogenic glycosides such as beta galactosidase acting on X-gal (bromocholoro indolyl galactoside). Proteases and peptidases can act on estersand amides such as N-acyl rhodamines. Another aspect of this inventionis the use of these other kinds of reactions as reporters of targets.Use of substrate analogs that are converted to reporting substrates ofthese enzymes in the presence of a target are further examples of themethods disclosed in the current invention.

For purposes of the methods of the present invention, useful carbocyclicmoieties have been described by Ferraro, M. and Gotor, V. in Chem Rev.2000, volume 100, 4319-48. Suitable sugar moieties are described byJoeng, L. S. et al., in J Med. Chem. 1993, vol. 356, 2627-38; by Kim H.O. et al., in J Med. Chem. 193, vol. 36, 30-7; and by Eschenmosser A.,in Science 1999, vol. 284, 2118-2124. Moreover, useful acyclic moietieshave been described by Martinez, C. I., et al., in Nucleic AcidsResearch 1999, vol. 27, 1271-1274; by Martinez, C. I., et al., inBioorganic & Medicinal Chemistry Letters 1997, vol. 7, 3013-3016; and inU.S. Pat. No. 5,558,91 to Trainer, G. L. Structures for these moietiesare shown below, where for all moieties R may be H, OH, NHR, F, N₃, SH,SR, OR lower alkyl and aryl; for the sugar moieties X and Y areindependently O, S, or NH; and for the acyclic moieties, X═O, S, NH, NR.

In certain embodiments, the sugar moiety in Formula I may be selectedfrom the following: ribosyl, 2′-deoxyribosyl, 3′-deoxyribosyl, 2′,3′-didehydrodideoxyribosyl, 2′,3′-dideoxyribosyl, 2′- or3′-alkoxyribosyl, 2′- or 3′-aminoribosyl, 2′- or 3′-fluororibosyl, 2′-or 3′-mercaptoribosyl, 2′- or 3′-alkylthioribosyl, acyclic, carbocyclicand other modified sugars.

Moreover, in Formula I, the base may include uracil, thymine, cytosine,5-methylcytosine, guanine, 7-deazaguanine, hypoxanthine,7-deazahypoxanthine, adenine, 7-deazaadenine, 2,6-diaminopurine oranalogs thereof.

Further, the terminal phosphate labeled nucleotides can be utilized withlabels which will not emit a signal unless and until the base isincorporated into the nucleotide, thereby releasing the polyphosphatewhich can then be acted on by an enzyme such as phosphatase to produce adetectable signal. In such systems, the label will undergo a chemicalchange from an inactive (non-detectable) form to an active (detectable)form when the base is incorporated, thereby providing information as tothe identity of the incorporated base. Since the color is only producedupon the reaction, the signal is generated against substantially nobackground, providing enhanced dynamic range of the assay. And, ifdesired, two or more differently labeled nucleotides can be added atonce permitting multiplexing of the assay.

The labels can be either chromophoric or fluorescent, as describedbelow, but are characterized in not necessarily comprising any quencher.Further, while the preferred use is in a nuicleotide sequencing orcharacterization assay as described herein, it is to be understood thatthese terminal phosphate labeled nucleotides can be conjugated to otherbinding molecules, such as antibodies or haptens, to permit homogeneousaffinity assay of these types to also be run in a multiplex format.

In the general assays, one or more terminally labeled nucleotides areplaced in the reaction mix and, if present, the complementary base isincorporated into the sequence by the nucleic acid polymerase; thiscauses a release of the dye labeled polyphosphate, which can then bedetected by the action of another enzyme, preferably a phosphatase, morepreferably shrimp alkaline phosphatase, calf intestine alkalinephosphatase, E. coli alkaline phosphatase or Rhodothermus marinusalkaline phosphatase. The label can then be detected by appropriatecolorimetric or fluorimetric means.

The label attached at the terminal-phosphate position in theterminal-phosphate-labeled nucleotide may be selected from the groupconsisting of 1,2-dioxetane chemiluminescent compounds, fluorogenicdyes, chromogenic dyes, mass tags and electrochemical tags. This wouldallow the detectable species to be detectable by the presence of any oneof color, fluorescence emission, chemiluminescene, mass change,electrochemical detection or a combination thereof.

Wherein the phosphorylated label in Formula I is a fluorogenic moiety,it is desirably selected from one of the following (all shown as thephosphomonester):2-(5′-chloro-2′-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone, soldunder the trade name ELF 97 (Molecular Probes, Inc.), fluoresceindiphosphate (tetraammonium salt), fluorescein3′(6′)-O-alkyl-6′(3′)-phosphate,9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (diammoniumsalt), 4-methylumbelliferyl phosphate (free acid), resorufin phosphate,4-trifluoromethylumbelliferyl phosphate, umbelliferyl phosphate,3-cyanoubelliferyl phosphate, 9,9-dimethylacridin-2-one-7-yl phosphate,6,8-difluoro-4-methylumbelliferyl phosphate and derivatives thereof.Structures of these dyes are shown below:

Wherein the phosphorylated label moiety in Formula I above is achromogenic moiety, it may be selected from the following:5-bromo-4-chloro-3-indolyl phosphate, 3-indoxyl phosphate, p-nitrophenylphosphate and derivatives thereof. The structures of these chromogenicdyes are shown as the phosphomonoesters below:

The moiety at the terminal-phosphate position may further be achemiluminescent compound wherein it is desired that it is aphosphatase-activated 1,2-dioxetane compound. The 1,2-dioxetane compoundmay include, but is not limited to, disodium2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′-(5-chloro-)tricyclo[3,3,1-1^(3,7)]-decan]-1-yl)-1-phenylphosphate, sold under the trade name CDP-Star (Tropix, Inc., Bedford,Mass.), chloroadamant-2′-ylidenemethoxyphenoxy phosphorylated dioxetane,sold under the trade name CSPD (Tropix), and3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane,sold under the trade name AMPPD (Tropix). The structures of thesecommercially available dioxetane compounds are disclosed in U.S. Pat.Nos. 5,582,980, 5,112,960 and 4,978,614, respectively, and areincorporated herein by reference.

The methods described above may further include the step of quantifyingthe nucleic acid sequence. In a related aspect, the detectable speciesmay be produced in amounts substantially proportional to the amount ofan amplified nucleic acid sequence. The step of quantifying the nucleicacid sequence is desired to be done by comparison of spectra produced bythe detectable species with known spectra.

In one embodiment, the invention provides a method of quantifying anucleic acid including the steps of: (a) conducting a nucleic acidpolymerase reaction, the polymerase reaction including the reaction of anucleotide which is substantially non-reactive to phosphatase inaddition to at least one terminal-phosphate-labeled nucleotide, whereinthe reaction results in the production of labeled polyphosphate; (b)permitting the labeled polyphosphate to react with a phosphatase toproduce a detectable by-product species in an amount substantiallyproportional to the amount of the nucleic acid to be quantified; (c)measuring the detectable species; and (d) comparing the measurementsusing known standards to determine the quantity of the nucleic acid. Inthis embodiment of the method of quantifying a nucleic acid, the nucleicacid to be quantified may be RNA. The nucleic acid may further be anatural or synthetic oligonucleotide, chromosomal DNA, or DNA.

The invention further provides a method of quantifying a DNA sequenceincluding the steps of: (a) conducting a DNA polymerase reaction in thepresence of a terminal-phosphate-labeled nucleotide wherein the reactionresults in the production of labeled polyphosphate; (b) permitting thelabeled polyphosphate to react with a phosphatase to produce adetectable by-product species in amounts substantially proportional tothe amount of the DNA sequence to be quantified; (c) measuring thedetectable species; and (d) comparing measurements using known standardsto determine the quantity of DNA. In this embodiment, the DNA sequencefor quantification may include natural or synthetic oligonucleotides, orDNA isolated from cells including chromosomal DNA.

In each of these methods of quantifying a nucleic acid sequencedescribed above, the polymerase reaction step may further includeconducting the polymerase reaction in the presence of a phosphatase. Asdescribed earlier in the specification, this would permit real-timemonitoring of nucleic acid polymerase activity and hence, real-timedetection of a target nucleic acid sequence for quantification.

The terminal-phosphate-labeled nucleotide useful for the methods ofquantifying the nucleic acid sequence provided herein may be representedby the Formula I shown above. The enzyme-activatable label becomesdetectable through the enzymatic activity of phosphatase which changesthe phosphate ester linkage between the label and the terminal-phosphateof a natural or modified nucleotide in such a way to produce adetectable species. The detectable species is detectable by the presenceof any one of or a combination of color, fluoresence emission,chemiluminescence, mass difference or electrochemical potential. Asalready described above, the enzyme-activatable label may be a1,2-dioxetane chemiluninescent compound, fluorescent dye, chromogenicdye, a mass tag or an electrochemical tag or a combination thereof.Suitable labels are the same as those described above.

As will be described in further detail in the Example Section, thepresent invention provides methods for determining the identity of asingle nucleotide in a target nucleic acid sequence. These methodsinclude the steps of: (a) conducting a nucleic acid polymerase reactionin the presence of at least one terminal phosphate-labeled nucleotide,which reaction results in the production of labeled polyphosphate; (b)permitting the labeled polyphosphate to react with a phosphatase toproduce a detectable species; (c) detecting the presence of thedetectable species; and (d) identifying the nucleoside incorporated. Indesired embodiments, the terminal phosphate-labeled nucleotide includesfour or more phosphates in the polyphosphate chain.

Another aspect of the invention relates to a nucleic acid detection kitincluding:

(a) at least one or more terminal-phosphate-labeled nucleotidesaccording to Formula I below:

wherein P=phosphate (PO₃) and derivatives thereof, n is 2 or greater; Yis an oxygen or sulfur atom; B is a nitrogen-containing heterocyclicbase; S is an acyclic moiety, carbocyclic moiety or sugar moiety; L isan enzyme-activatable label containing a hydroxyl group, a sulfliydrylgroup or an amino group suitable for forming a phosphate ester, athioester or a phosphoramidate linkage at the terminal phosphate of anatural or modified nucleotide; P-L is a phosphorylated label whichpreferably becomes independently detectable when the phosphate isremoved.

-   -   (b) at least one of DNA polymerase, RNA polymerase or reverse        transcriptase; and    -   (c) phosphatase.

The sugar moiety in the terminal-phosphate-labeled nucleotide includedin the kit may include, but is not limited to ribosyl, 2′-deoxyribosyl,3′-deoxyribosyl, 2′, 3′-dideoxyribosyl, 2′, 3′-didehydrodideoxyribosyl,2′- or 3′-alkoxyribosyl, 2′- or 3′-aminoribosyl, 2′- or3′-fluororibosyl, 2′- or 3′-mercaptoribosyl, 2′- or 3′-alkylthioribosyl,acyclic, carbocyclic and other modified sugars.

The base may be, but is not limited to uracil, thymine, cytosine,5-methylcytosine, guanine, 7-deazaguanine, hypoxanthine,7-deazahypoxanthine, adenine, 7-deazaadenine and 2,6-diaminopurine andanalogs thereof.

Furthermore, as described above, the enzyme-activatable label may be a1,2-dioxetane chemiluminescent compound, fluorescent dye, chromogenicdye, a mass tag, an electrochemical tag or a combination thereof.Suitable compounds for conjugation at the terminal-phosphate position ofthe nucleotide are the same as those described above.

EXAMPLES Example 1

Preparation of γ-(4-trifluoromethylcoumarinyl)ddGTP (γCF₃Coumarin-ddGTP)

ddGTP (200 ul of 46.4 mM solution, purity>96%) was coevaporated withanhydrous dimethylformamide (DMF, 2×0.5 ml). To thisdicyclohexylcarbodiimide (DCC, 9.6 mg, 5 eq.) was added and mixture wasagain coevaporated with anhyd. DMF (0.5 ml). Residue was taken in anhyd.DMF (0.5 ml) and mixture was allowed to stir overnight. There was stillca 20% uncyclized triphosphate (could be from hydrolysis of cyclictrimetaphosphate on the column). To the mixture another 2 eq. of DCC wasadded and after stirring for 2 h, 7-hydroxy-4-trifluoromethyl coumarin(4-trifluoromethylumbelliferone, 42.7 mg, 20 eq.) and triethylamine (26ul, 20 eq.) were added and mixture was stirred at RT. After 2 days, HPLC(0-30% acetonitrile in 0.1M triethylammonium acetate (TEAA) in 15minutes, 30-50% acetonitrile in 5 min and 50-100% acetonitrile in 10minutes, C18 3.9×150 mm column, flow rate 1 ml/minute) showed a newproduct at 9.7 min and starting cyclic triphosphate (ratio of 77 to 5 at254 nm). Mixture was allowed to stir for another day. P-31 NMR showedgamma labeled nucleoside-triphosphate as the main component of reactionmixture. Reaction mixture was concentrated on rotary evaporator. Residuewas extracted with water (5×1 ml). HPLC showed a purity of 82% at 254 nmand 81% at 335 nm. Combined aq solution was conc. on rotary evaporatorand redissolved in water (1 ml). It was purified on 1 inch×300 cm C18column using 0-30% acetonitrile in 0.1M triethylammonium bicarbonate(TEAB, pH 8.3) in 30 min and 30-50% acetonitrile in 10 min, 15 ml/minflow rate. Product peak was collected in 3 fractions. Fraction 1 wasrepurified using the same preparative HPLC method as above except the pHof the TEAB buffer was reduced to 6.7 by bubbling CO₂. Product peak wasconcentrated and coevaporated with MeOH (2 times) and water (1 time).Sample was dissolved in 1 ml water. HPLC showed a purity of>99% at 254and 335 nm. UV showed a conc. of 2.2 mM assuming an extinction coeff. of11,000 at 322 nm (reported for beta galactoside derivative of7-hydroxy-4-trifluoromethylcoumarin, Molecular Probes Catalog). MS:M⁻=702.18 (calc 702.31), UV γ_(A)=253, 276 & 322 nm. Thetrifluorocoumarin dye attached to the gamma phosphate of ddGTP isfluorescent with an excitation maximum of 322 nm and an emission maximumof about 415 nm. Upon hydrolysis of the phosphate ester to release thefree coumarin dye, the spectrum changes with excitation maximum of about385 nm and emission maximum of about 502 nm. This change is readilydetected by simple fluorescence measurements or color change. Synthesisof gamma nucleotides has been generally described by Arzumanov, A. etal. in J Biol Chem (1996) October 4; 271 (40): 24389-94.

Example 2

Preparation of γ-(3-Cyanocoumarinyl)ddATP (γCNCoumarin-ddATP)

ddATP (100 μl of 89 mM solution, >96%) was coevaporated with anhydrousDMF (2×1 ml). To this DCC (9.2 mg, 5 eq.) was added and mixture wasagain coevaporated with anhydrous DMF (1 ml). Residue was taken inanhydrous DMF (0.5 ml) and reaction was stirred at rt. After overnight7-hydroxy-3-cyanocoumarin (33.3 mg, 20 eq.) and TEA (25 ul, 20 eq.),were added and mixture was stirred at RT. After 1 day, a major product(55% at 254 nm) was observed 8.1 min with another minor product at 10min (˜10%). No significant change occurred after another day. Reactionmixture was concentrated on rotary evaporator and residue was extractedwith 3×2 ml water and filtered. Aq solution was concentrated andpurified on C-18 using 0-30% acetonitrile in 0.1M TEAB (pH 6.7) in 30min and 30-50% acetonitrile in 10 min, flow rate 15 ml/min. Main peakwas collected in 3 fractions. HPLC of the main peak (fr. 2) showed apurity of 95.6% at 254 nm and 98.1% at 335 nm. It was concentrated onrotary evaporator (at RT), coevaporated with MeOH (2×) and water (1×).Residue was dissolved in 0.5 ml water. A 5 ul sample was diluted to 1 mlfor UV analysis. A346 nm=0.784. Assuming an extinction coeff. of 20,000(reported for 7-ethoxy-3-cyanocoumarin, Molecular Probes Catalog),concentration=7.84 mM. Yield=3.92 umol, 44%. Sample was repurified onC-18 column using same method as above. Sample peak was collected in 3fractions. Fractions 2 & 3, with >98% purity at 254 nm and >99.5% purityat 340 nm, were combined. After concentration, residue was coevaporatedwith MeOH (2×) and water (1×). Sample was dissolved in water (1 ml) togive a 2.77 mM solution. MS: M⁻=642.98 au (calc 643.00 au), UV γ_(A)=263& 346 nm The cyanocoumarin dye attached to the gamma phosphate of ddATPis fluorescent with an excitation maximum of 346 nm and an emissionmaximum of about 411 nm. Upon hydrolysis of the phosphate ester torelease the free coumarin dye, the spectrum changes with excitationmaximum of about 408 nm and emission maximum of about 450 nm. Thischange is readily detected by simple fluorescence measurements or colorchange. Synthesis of gamma nucleotides has been generally described byArzumanov, A, et al in J Biol Chem. (1996) October 4;271(40):24389-94.

Example 3

Preparation ofδ-9H(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)-dideoxythymidine-5′-tetraphosphate(ddT4P-DDAO)

ddTTP (100 μl of 80 mM solution) was coevaporated with anhydrousdimethylformamide (DMF, 2×1 ml). To this dicyclohexylcarbodimide (8.3mg. 5 eq.) was added and the mixture was again coevaporated withanhydrous DMF (1 ml). Residue was taken in anhydrous DMF (1 ml) andreaction was stirred at room temperature overnight. HPLC showed mostlycyclized triphosphate (˜82%). Reaction mixture was concentrated andresidue was washed with anhydrous diethyl ether 3×. It was redissolvedin anhydrous DMF and concentrated to dryness on rotavap. Residue wastaken with DDAO-monophosphate, ammonium salt (5 mg, 1.5 eq.) in 200 μlanhydrous DMF and stirred at 40° C. over the weekend. HPLC showedformation of a new product with desired UV characteristics at 11.96 min.(HPLC Method: 0.30% acetonitrile in 0.1M triethylammonium acetate (pH 7)in 15 min, and 30-50% acetonitrile in 5 min, Novapak C-18 3.9×150 mmcolumn, 1 ml/min). LCMS (ES−) also showed a major mass peak 834 for M−1peak. Reaction mixture was concentrated and purified on Deltapak C18,19×300 mm column using 0.1M TEAB (pH 6.7) and acetonitrile. Fractionwith product was repurified by HPLC using the same method as describedabove. Fraction with pure product was concentrated, coevaporated withMeOH (2×) and water (1×). Residue was dissolved in water (1.2 ml) togive a 1.23 mM solution. HPCL purity as 254 nm>97.5%, at 455 nm>96%; UVγ_(A)=267 nm and 455 nm; MS: M−1=834.04 (calc 8.33.95).

δ-9H(1,3-dichloro-9,9-dimethylacridin-2-one-7=yl)-dideoxycytidine-5′-tetraphosphate(ddC4P-DDAO),δ-9H(1,3-dichloro-9,9-dimethylacridin-2-one-dideoxyadenosine-5′-tetraphosphate(ddA4P-DDAO) andδ-9H(1,3-dichloro-9,9-dimethylacridin-2-one-y-YL)-dideoxyguanosine-5′-tetraphosphate(ddG4P-DDAO) were synthesized and purified in a similar fashion.Analysis of these purified compounds provided the following data:ddC4P-DDAO: UV γ_(A)=268 nm and 454 nm; MS: M−1=819.32 (calc 818.96);ddA4P-DDAO: UV γ_(A)=263 nm and 457 nm; MS: M−1=843.30 (calc 842.97);ddG4P-DDAO: UV γ_(A)=257 nm and 457 nm; MS: M−1=859.40 (calc 858.97).

Example 4

Preparation of ε-9H(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)-dideoxythymidine-5′-pentaphosphateDDAO-ddT-pentaphosphate (ddT5P-DDAO)

A. Preparation of DDAO Pyrophosphate

DDAO-phosphate diammonium salt (11.8 umol) was coevaporated withanhydrous DMF (3×0.25 ml) and was dissolved in DMF (0.5 ml). To thiscarbonyldiimidazole (CDI, 9.6 mg, 5 eq) was added and the mixture wasstirred at room temperature overnight. Excess CDI was destroyed byaddition of MeOH (5 ul) and stirring for 30 minutes. To the mixturetributylammoniumdihydrogen phosphate (10 eq., 236 ml of 0.5 M solutionin DMF) was added and the mixture was stirred at room temperature for 4days. Reaction mixture was concentrated on rotavap. Residue was purifiedon HiPrep 16.10 Q XL column using 0-100% B using 0.1M TEAB/acetonitrle(3:1) as buffer A and 1 M TEAB/acetonitrile (3:1) as buffer B. Main peak(HPLC purity 98%) was collected, concentrated and coevaporated withmethanol (2×). Residue was dissolved in 1 ml water to give 5.9 mMsolution. UV/VIS γ_(max)=456 nm.

B. Preparation of ddT5P-DDAO

ddTTP (100 ul of 47.5 mM solution in water) was coevaporated withanhydrous DMF (2×1 ml). To this DCC (5 eq., 4.9 mg) was added andmixture was coevaporated with DMF (1×1 ml). Residue was taken inanhydrous DMF (0.5 ml) and stirred at room temperature for 3 hours. Tothis 1.03 eq of DDAO pyrophosphate, separately coevaporated withanhydrous DMF (2×1 ml) was added as a DMF solution. Mixture wasconcentrated to dryness and then taken in 200 ul anhydrous DMF. Mixturewas heated at 38° C. for 2 days. Reaction mixture was concentrated,diluted with water, filtered and purified on HiTrap 5 ml ion exchangecolumn using 0-100% A-B using a two step gradient. Solvent A=0.1MTEAB/acetonitrile (3:1) and solvent B=1M TEAB/acetonitrile (3:1).Fraction 12×13 which contained majority of product were combined,concentrated and coevaporated with methanol (2×). Residue was repurifiedon Xterra RP C-18 30-100 mm column using 0.30% acetonitrile in 0.1M TEABin 5 column and 30-50% acetonitrile in 2 column volumes, flow rate 10ml/min. Fraction containing pure product was concentrated andcoevaporated with methanol (2×) and water (1×). HPLC purity at 455nm>99%. UV/VIS=268 nm and 455 nm. MS: M−1=914.03 (calc 913.93).

The DDAO dye attached to the gamma phosphate of these polyphosphates isfluorescent with an excitation maximum of 455 nm and an emission maximumof about 608 nm. Upon hydrolysis of the phosphate ester to release thefree dye, the spectrum changes with excitation maximum of about 645 nmand emission maximum of about 659 nm. The change is readily detected bysimple fluorescence measurements or color change.

It is noted that similar nucleotide compounds with dyes or otherdetectable moieties attached to the terminal phosphate could also bemade using similar methods to those described in Examples 1-4 above.These include ribonucleotides, deoxyribonucleotides,nucleoside-tetraphosphates, nucleotides with any of thenaturally-occurring bases (adenine, guanine, cytosine, thymine,hypoxanthine and uracil) as well as modified bases or modified sugars.

Example 5

Preparation of Ethyl-Fluorescein Triphosphate

Ethyl-fluorescein (100 mg) was coevaporated with anhydrous acetonitrile(2 times) and resuspended in anhydrous acetonitrile (5 ml). To thisphosphoryl chloride (78 ul) was added. After stirring at 0° C. for 30minutes, three equivalents of pyridine was added. Mixture was allowed towarm to room temperature and stirred at room temperature for 3 hours. Tothe reaction mixture, tributylammonium pyrophosphate in DMF (0.5 M, 10equivalents) and tributylamine (15 equivalents) were added. Afterstirring for 5 minutes, reaction was quenched with 15 ml of 1Mtriethylammonium bicarbonate. Reaction mixture was concentrated andcoevaporated with methanol (2 times). Residue was purified byion-exchange chromatography on a HiPrep 16×10 Q XL column followed by areverse phase chromatography on Xterra C-18 30×100 mm column to yield37.2 umol of pure product with a lamdamax at 274 nm. M−1=598.99 (calc.599). Proton and phosphorous NMR spectra corresponded toethyl-fluorescein phosphate of structure shown below.

Examples 6 and 7 below demonstrate that dideoxynucleotides having a dyederivative attached to the terminal phosphate may be effectivelyincorporated as substrates into a growing nucleic acid chain by anucleic acid polymerase in a template-directed process for detection ofa nucleic acid.

Example 6

Nucleic Acid Sequence Detection Using Polymerase Incorporation of GammaPhosphate-labeled ddGTP

Reactions were assembled at room temperature (23° C.) using thedideoxynucleotide of Example (1). Reactions contained primer templatecombinations having a single oligonucleotide primer (represented by SEQID NO: 1) annealed to one of two different oligonucleotide templateswith either a dC or a dT as the next template nucleotide adjacent the 3′terminus of the primer, corresponding to SEQ ID NO: 2 and SEQ ID NO: 3,respectively.

Referring now to FIG. 1, for template 1 (SEQ ID NO: 2) in the presentexample, DNA polymerase would be expected to extend the primer withlabeled ddGTP. Similarly, for template 2 (SEQ ID NO: 3) in FIG. 1, DNApolymerase would be expected to extend the primer with ddATP, but notwith labeled ddGTP.

Reaction conditions: A 70 μl reaction containing 25 mM Tris, pH 8.0, 5%glycerol 5 mM MgCl₂, 0.5 mM beta-mercaptoethanol, 0.01% tween-20, 0.25units shrimp alkaline phosphatase, 100 nM primer annealed to template(the next template nucleotide is either dCMP or dTMP, as indicated), and2 μM ddGTP-CF₃-Coumarin was assembled in a quartz fluorescenceultra-microcuvet in a LS-55 Luminescence Spectrometer (Perkin Elmer),operated in time drive mode. Excitation and emission wavelengths are 390nm and 500 nm respectively. Slit widths were 5 nm for excitation slits,15 nm for emission slits. The reaction was initiated by the addition of0.35 μl (11 units) of a cloned DNA polymerase I genetically engineeredto eliminate 3′-5′ exonuclease activity, 5′-3′ exonuclease activity anddiscrimination against dideoxynucleotides and 0.25 mM MnCl₂.

As shown in FIG. 1, for reactions containing the gamma labeled ddGTP,dye emission was detected only with Primer: Template 1, where the nextnucleotide in the template was a dC. Cleavage of the pyrophosphateproduct of phosphoryl transfer by shrimp alkaline phosphatase leads to adetectable change in the CF₃-coumarin label which allows for thedetection of the nucleic acid. No detectable dye emission was obtainedwith Primer: Template 2.

Example 7

Nucleic Acid Sequence Detection Using Polymerase Incorporation of GammaPhosphate-Labeled ddATP

Reactions were assembled at room temperature (23° C.) using thedideoxynucleotide of Example (2). Reactions contained primer: templatecombinations having a single oligonucleotide primer (SEQ ID NO: 1)annealed to one of two different oligonucleotide templates with either adC or a dT as the template nucleotide, adjacent to the 3′ terminus ofthe primer, corresponding to SEQ ID NO: 2 and SEQ ID NO: 3,respectively.

Referring now to FIG. 2, for template 2 (SEQ ID NO: 3) in the presentexample, DNA polymerase would be expected to extend the primer withlabeled ddATP. Similarly, for template 1 (SEQ ID NO: 3) in FIG. 2, DNApolymerase would be expected to extend the primer with ddGTP, but notwith labeled ddATP.

Reaction conditions: A 70 μl reaction containing 25 mM Tris, pH 8.0, 5%glycerol 5 mM MgCl₂, 0.5 mM beta-mercaptoethanol, 0.01% tween-20, 0.25units shrimp alkaline phosphatase, 100 nM primer annealed to template,and 2 μM ddATP-CN-Coumarin was assembled in a quartz fluorescenceultra-microcuvet in a LS-55 Luminescence Spectrometer (Perkin Elmer),operated in time drive mode. Excitation and emission wavelengths are 410nm and 450 nm respectively. Slit widths were 5 nm for excitation slits,15 nm for emission slits. The reaction was initiated by the addition of0.35 μl (11 units) of a cloned DNA polymerase I genetically engineeredto eliminate 3′-5′ exonuclease activity, 5′-3′ exonuclease activity anddiscrimination against dideoxynucleotides and 0.25 mM MnCl₂.

As shown in FIG. 2, for reactions containing the gamma labeled ddATP,dye emission was detected only for Primer: Template 2, where the nextnucleotide in the template was a dT. Cleavage of the pyrophosphateproduct of phosphoryl transfer by shrimp alkaline phosphatase produces adetectable change in the CN-coumarin label that allows one to detect thenucleic acid. No detectable dye emission was obtained with Primer:Template 1.

1. A method of detecting a target using a labeled substrate or substrateanalog comprising reacting said substrate or substrate analog with saidtarget in an enzyme-catalyzed reaction to produce a labeledpolyphosphate that has independently detectable signal only when saidsubstrate or substrate analog reacts, and wherein, when the target is anucleic acid, the labeled polyphosphate generated from theenzyme-catalyzed reaction has three or more phosphates.
 2. The method ofclaim 1 wherein the labeled polyphosphate can further undergo a chemicalchange, to produce the detectable signal.
 3. The method of claim 2wherein the chemical change is catalyzed by one or more enzymes toproduce the detectable signal.
 4. The method of claim 1 wherein thetarget is a nucleic acid target.
 5. The method of claim 1 wherein thetarget is a protein.
 6. The method of claim 1 wherein labeled substrateanalog is a terminal-phosphate labeled nucleoside polyphosphate.
 7. Themethod of claim 1 wherein the said enzyme-catalyzed reaction is nucleicacid polymerization.
 8. The method of claim 1 wherein the saidenzyme-catalyzed reaction is catalyzed by a phosphodiesterase.
 9. Themethod of claim 1 wherein the said enzyme-catalyzed reaction iscatalyzed by a dinucleotide phosphorylase.
 10. The method of claim 1wherein the said enzyme-catalyzed reaction is catalyzed by a ligase. 11.The method of claim 1 wherein the label in said labeled polyphosphate,is a colorimetric, a fluorescent, a chromogenic, a fluorogenic or achemiluminescent compound or an electrochemical label.
 12. The method ofclaim 2 wherein the said label is selected from the group consisting ofchemiluminescent compounds, fluorogenic dyes, chromogenic dyes,electrochemical tags and combinations thereof.
 13. The method of claim 1wherein said detectable species is detectable by a property selectedfrom the group consisting of color, fluorescence emission,chemiluminescence, mass change, reduction/oxidation potential andcombinations thereof.
 14. The method of claim 11 wherein the saidcolorimetric label is selected from the group consisting of cyanines,merrocyanines, phenoxazines, acridinones or nitrophenols.
 15. The methodaccording to claim 11 wherein the said fluorescent label is selectedfrom fluoresceins, rhodamines, cyanines or merrocyanines.
 16. The methodof claim 12 wherein said label is a fluorogenic moiety is selected fromthe group consisting of2-(5′-chloro-2′-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone,fluorescein diphosphate, fluorescein 3′(6′)-O-alkyl-6′(3′)-phosphate,9H-(1,3 -dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate,4-methylumbelliferyl phosphate, resorufin phosphate,4-trifluoromethylumbelliferyl phosphate, umbelliferyl phosphate,3-cyanoumbelliferyl phosphate, 9,9-dimethylacirdin-2-one-7-yl phosphate,6,8-difluoro-4-methylumbelliferyl phosphate, and derivatives thereof.17. The method of claim 12 wherein said label is a chromogenic moietyselected from the group consisting of 5-bromo-4-chloro-3-indolylphosphate, 3-indoxyl phosphate, p-nitrophenyl phosphate and derivativesthereof.
 18. The method of claim 12 wherein said chemiluminescentcompound is a phosphatase-activated 1,2-dioxetane compound.
 19. Themethod of claim 18 wherein said 1,2-dioxetane compound is selected fromthe group consisting of2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′-(5-chloro-)tricyclo[3,3,1-1^(3,7)]-decan]-1-yl)-1-phenylphosphate, chloroadamant-2′-ylidenemethoxyphenoxy phosphorylateddioxetane,3-(2′-spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2-dioxetaneand derivatives thereof.
 20. The method of claim 3 wherein the saidenzyme is selected from a phosphatase, a glycosidase, a polyphosphatetransferring enzyme, a peptidase, an oxidase, a peroxidase, a sulfatase,an esterase or a combination thereof.
 21. The method of claim 2 whereinsaid chemical change is caused by chemical hydrolysis.
 22. The method ofclaim 2 wherein the said chemical change is caused by a combination ofchemical hydrolysis and enzymatic action.
 23. The method according toclaim 22 wherein the chemical hydrolysis is spontaneous.
 24. A method ofdetecting a target nucleic acid sequence using a labeled nucleotideanalog comprising incorporating said analog by a nucleic acid polymeraseinto a polynucleotide complimentary to the target sequence thereby,producing a species which is a labeled polyphosphate with three or morephosphate groups that undergoes a further chemical change to produce adetectable signal.
 25. The method of claim 22 wherein said chemicalchange is catalyzed by one or more enzymes.
 26. The method of claim 22wherein the said species is represented by the formulaLabel-x-p-(p)_(n)-p wherein Label is a detectable moiety, x is a O, S,NH or a linker, n=1-4 and p is a phosphate or phosphate derivative. 27.The method of claim 26 wherein the linker is an enzyme-removable linker.28. The method according to claim 26 wherein the linker is spontaneouslyremoved after removal of phosphate.